CC BY
3References
1. Coronavirus disease (COVID-19) Pandemic. Geneva: World Health Organization, March 23, 2020. https://www.who.int/emergencies/diseases/novelcoro-navirus-2019
2. WHO. Coronavirus disease (COVID-19) Pandemic. Geneva:World Health Organization, March 23, 2020
(https://www.who.int/emergencies/diseases/novel-coronavirus-2019).
3. Schwartz B.J. New Poll: COVID-19 Impacting Mental WellBeing: Americans Feeling Anxious, Especially for Foved Ones.APA News releases. March 25, 2020. https://www.psychiatry.org/new sroom /new s-releases/new -poll-covid- 19-impactingmentalwell-be-ing-americans-feeling-anxious-especially-for-lovedones-older-adults-are-lessanxious.
4. Fuhby T. CNN, Published online April 16, 2020 https://edition.cnn.com/2020/04/16/health/anti-anxiety-medication-usdemandcoronavirus/index.html.
5. Brooks SK, Webster RK, Smith FE, Woodland F, Wessely S,Greenberg N, Rubin GJ. The psychological impact of quarantineand how to reduce it: rapid review of the evidence. Fancet.
2020;395(10227):912-920. https://doi.org/10.10l6/s0140-6736(20)30460-8.
6. Fondon RT. Is COVID-19 leading to a mental illnesspandemic? Presented by ID Practitioner in MDedge Infectiousdisease. Published online March 25,2020. https://www.mdedge. com /infectiousdis-ease/article/219612/coronavirus-updates/covid-
191eading-mental-illness-pandemic?fbclid
7. Galea S, Merchant RM, Furie N.The Mental Health Consequences of COVID-19 and Physical Distancing: The Need for Prevention and Early Intervention. JAMA Intern Med. Published online April 10.2020. https://doi.Org/10.1001/jamaintern-med.2020.1562.
8. Rossi R, Socci V, Talevi D, Mensi S, Niolu C, Pacitti F, Di Marco A, Rossi A, Siracusano A, Di Fo-renzo G. (). COVID-19 pandemic andlockdown measures impact on mental health among the generalpopulation in Italy. An N=18147 web-based survey. 2020. medRxivpreprint doi: https://doi.Org/10.1101/2020.04.09.20057802
9. Galea S, Merchant RM, Lurie N. The Mental Health Consequences of COVID-19 and Physical Distancing: The Need for Prevention and Early Intervention. JAMA Intern Med. Published online April 10, 2020.https://doi.org/10.1001/jamainternmed.2020.156 2
10. Кекелидзе З.И., Чехонин В.П. Критические состояния впсихиатрии. Клинические и иммунохи-мические аспекты. М.:Государственный научный центр социальной и судебной психиатрии им. В.П. Сербского; 1997, 362 с.
11. Александровский Ю.А., Чехонин В.П. Клиническая иммунология пограничных психических расстройств М.: ГЭОТАРМедиа; 2005, 235 с.
12. Мосолов C.H., Малин Д.И., Цукарзи Э.Э., Костюкова Е.Г. Особенности психофармакотерапии пациентов с коронавирусной инфекцией (COVID-19). Современная терапия психических расстройств. 2020: 2. https://doi.org/10.21265/PSYPH.2020.18.69.001
EVALUATION OF THE EFFECTIVENESS OF THE USE OF AUTOLOGOUS MULTIPOTENT MESENCHYMAL CELLS TRANSDIFFERENTIATED INTO CHONDROBLASTS IN THE
EXPERIMENT
Korotkov A.V.,
Candidate of Medical Sciences, Associate Professor, Ural State Medical University 620028, st. Repin, 3, Yekaterinburg, Russia, Institute of Medical Cell Technologies, 620026, st. Karl Marx, 22 a, Yekaterinburg, Russia
Sichkar D.A.,
Senior Lecturer Ural State Medical University 620028, st. Repin, 3, Yekaterinburg, Russia, Institute of Medical
Cell Technologies, 620026, st. Karl Marx, 22 a, Yekaterinburg, Russia
Yakovleva E.A.,
research trainee Ural State Medical University 620028, st. Repin, 3, Yekaterinburg, Russia
Shuman E.A.,
Senior Lecturer Ural State Medical University 620028, st. Repin, 3, Yekaterinburg, Russia, Institute of Medical
Cell Technologies, 620026, st. Karl Marx, 22 a, Yekaterinburg, Russia
Satonkina O.A.,
Candidate of biological sciences, Associate Professor, Ural State Medical University 620028, st. Repin, 3, Yekaterinburg, Russia, Institute of Medical Cell Technologies, 620026, st. Karl Marx, 22 a, Yekaterinburg, Russia
Makeev O.G.
Professor, doctor of medical sciences Ural State Medical University 620028, st. Repin, 3, Yekaterinburg, Russia, Institute of Medical Cell Technologies, 620026, st. Karl Marx, 22 a, Yekaterinburg, Russia
Abstract
Objective: to evaluate the efficiency of using chondroblast-differentiated multipotent mesenchymal stromal cells (CMMSC) in the treatment of hyaline cartilage damage on a model of a defect in the articular surface of the rabbit knee joint.
Material and methods. the study included 10 chinchilla rabbits. The first group of animals (5 individuals) had a full-thickness defect in the cartilage of the loaded zone of the patellofemoral articulation of the left femur. the
second group of animals (5 individuals) formed a partial-thickness cartilage defect in the above zone. After suturing the wound, 2x106 HMMSC were injected into the joint cavity to create a control group for each animal, a defect in the cartilage of the contralateral knee joint was formed, after which 0.5 ml of 0.9% sodium chloride solution was injected intra-articularly. 11 weeks after the operation, a macroscopic and microscopic examination of the areas of cartilage defect was carried out with an assessment of the characteristics of the formed tissue using the O'Driscoll scale.
Results. The group of animals, in the treatment of a full-thickness cartilage defect of which CMSC were used, was characterized by significantly higher indicators of the categories "tissue morphology", "staining of the extracellular matrix", "Structure integrity", "thickness of the formed cartilage", "Cluster of chondrocytes", "severity of inflammation" (p <0.05) and the final value of the O'Driscoll scale (p <0.01) compared with the control. in the group of animals with a partial defect in the cartilage of the knee joint after the application of CMSC, higher values of indicators of the categories "severity of inflammation", "hypocellularity" (p <0.05) and the final value of the O'Driscoll scale (p <0.05) were also noted. compared with control. we did not reveal any complications with intraarticular injection of CMSC.
Conclusion. Intra-articular injection of ChMMSC has a stimulating effect on the regeneration of hyaline cartilage in both full-thickness and partial-thickness defects, improves the quantitative and qualitative characteristics of the tissue newly formed in the process of regeneration, and is a safe method of treating traumatic defects of the cartilage of the articular surfaces.
Keywords: knee joint, cartilage defect, chondroblasts
Introduction
Damage to the cartilage of the joints is quite common. So, according to arthroscopic data, this pathology is detected in 44-66% of cases [1]. Hyaline cartilage has an extremely limited potential for spontaneous regeneration, which is associated with the low ability of chondrocytes to proliferate, their insufficient mobility, and the absence of vascularization of the extracellular matrix [2]. Even small chondromalacia can induce further progressive damage to the surrounding cartilage defect and lead to early manifestation of osteoarthritis, persistent limitation of joint function and a decrease in the quality of life of patients [3].
Treatment of this pathology is a difficult and still unsolved problem. For the purpose of conservative therapy of patients, non-steroidal anti-inflammatory drugs, analgesics, chondroprotectors, and the introduction of steroid hormones into the joint cavity are used. Despite the rather extensive arsenal of drugs and various physiotherapeutic techniques, unfortunately, their use only reduces the severity of symptoms and does not prevent further progressive damage to hyaline cartilage [4].
The ineffectiveness of conservative treatment of this pathology has determined the emergence of a large number of various surgical interventions. Among them, surgical techniques are widely used, aimed at stimulating reparative processes in the hyaline cartilage due to the stem cells of the bone marrow of the subchondral layer. These methods include antegrade and retrograde tunneling of the subchondral bone, microfracturing, "debrigment". The clinical efficacy of osteoperforative techniques is still ambiguously assessed by various researchers. Many authors associate insufficiently good long-term results with the formation of fibrocartilaginous tissue in the defect zone, which is functionally inferior to hyaline cartilage [4]. However, a number of researchers have established that the mechanical properties of cartilage tissue formed from mesenchymal stem cells, its ultrastructure, and the composition of the extracellular matrix are superior in their characteristics to the tissue formed during regeneration by young or
mature chondrocytes [5]. In recent years, there have been reports of good long-term results of the use of osteoperforative techniques in the treatment of hyaline cartilage injuries, especially among young patients [6].
In surgical treatment, various types of osteochondral autotransplantation and chondrocyte transplantation are also used to replace articular cartilage defects. However, these techniques are also not without drawbacks. Thus, the indications for osteochondral autotransplantation are limited, and the procedure itself is associated with the risk of early development of osteoarthritis in the patellofemoral joint [4]. Chondrocyte autotransplantation is associated with significant material costs, requires expensive equipment and highly qualified personnel for the cultivation of cell material. Despite the rather high level of development of biotechnology, the result of autotransplantation of chondrocytes is not always the formation of hyaline cartilage tissue in the defect zone [7].
In recent decades, cell therapy has taken an increasingly strong position in clinical medicine. In this regard, the possibility of using MMSC differentiated into chondroblasts in the treatment of patients with hyaline cartilage lesions of the articular surfaces is of great interest. The presence of growth factors in CMMSC, which provide the processes of regeneration and hemostasis, are the basis for its use. CMMSK contains not only growth factors (PDGF - platelet growth factor, TGF-p - transforming growth factor, EGF - epithelial growth factor, VEGF - vascular endothelial growth factor), but also adhesive molecules (fibrin, fi-bronectin and vitronectin) necessary for migration, cell adhesion and stimulation of collagen synthesis. CMSCs also produce cytokines with an anti-inflammatory effect, stimulating repair and anabolic processes in damaged tissues. Since CMSC is a derivative of the patient's own cells, their use is not associated with the risk of parenteral transmission of infections such as HIV or hepatitis. The use of CMSC does not cause hyperplastic processes, carcinogenesis or tumor growth. Growth factors are not mutagens and do not block the feedback
mechanisms of the processes of tissue repair and regeneration [10].
ChMMSC is widely used in traumatology, orthopedics and sports medicine in the treatment of enthe-sopathies, injuries of the rotator cuff of the shoulder, atrophic pseudoarthrosis, large bone cysts and associated pathological fractures, injuries of fingers with skin and soft tissue defects [11].
In recent years, researchers have obtained good results when using CMMSC in the treatment of osteoarthritis [12]. Although these studies suggest that CMSC has chondroprotective properties, its effect on regeneration processes in damaged articular cartilage remains insufficiently studied to date. There are also no data on the effectiveness of intra-articular administration of CMMSC in the treatment of post-traumatic defects in hyaline cartilage.
The aim of this study was to evaluate the effectiveness of the use of chondroblast-differentiated MMSCs in the treatment of hyaline cartilage injuries using a model of a defect in the articular surface of the rabbit knee joint.
Material and methods
The study was conducted in compliance with the requirements of humane treatment of experimental animals and complies with the recommendations of the
Council of Europe Convention on the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes.
Characteristics of animals In the experiment were taken 10 rabbits of both sexes of the chinchilla breed weighing 2400-3500, the Animals were kept in a vivarium in accordance with international GLP rules. The animals were kept under quarantine for at least 14 days. Modeling of joint pathology The rabbit was fixed on a special manipulation table. After preparation of the operating field, a medial parapatellar approach to the left knee joint was performed. a cartilage defect with a diameter and depth of 3 mm in the loaded zone of the patellofemoral articulation of the femur in 5 animals (study group 1) was formed with a trephine of the corresponding diameter until bleeding from the subchondral bone appeared (Fig. 1 A).
In the other 5 animals, a partial-thickness defect in the cartilage of the left knee joint was formed (Fig. 1B) in the loaded zone of the patellofemoral articulation of the femur, 3 mm in diameter and 1 mm in depth (study group 2).
m
FIG. 1.
Formed defect in the cartilage of the knee joint of the rabbit. A - full layer defect, B incomplete layer defect.
After the completion of the manipulations, the surgical wound was sutured in layers without drainage, then 0.5 ml of CMSC was injected into the cavity of the knee joint using an injection needle.
To create a control group, each animal was formed a defect in the cartilage of the right knee j oint according to the above technique (full-layer and non-full-layer for animals from groups 1 and 2, respectively), after which 0.5 ml of 0.9% sodium chloride solution was injected intra-articularly.
In the postoperative period, for the prevention of pyoinflammatory complications, the animals were injected intramuscularly with 10 mg / kg of cefazolin once. Limb immobilization was not performed.
After 11 weeks from the moment of the operation, the animals were withdrawn from the experiment. Euthanasia was performed by air embolism under intravenous anesthesia. Then, a macroscopic and microscopic assessment of the regeneration processes in the zone of defects in the knee joint cartilage was carried out. mac-
roscopic examination assessed the nature of the articular surface in the defect zone and signs of gonarthrosis development. For histological examination, the distal femur was fixed in 10% formalin solution, then decalcified in 5% nitric acid solution. After standard wiring, the joint area with the cartilage defect zone was enclosed in paraffin blocks, after which sagittal sections with a thickness of 5 ^m were prepared. the obtained sections were stained with hematoxylin and eosin and examined under a light microscope "Leica DM 2000" at 100 and 200 times magnification. The O'Driscoll scale was used to assess the characteristics of tissue formed during regeneration [13].
The nature of the distribution of the data obtained as a result of the study was checked using the Shapiro-Wilk test. Data with distribution other than normal were presented as median and percentiles. The MannWhitney U-test was used to assess the differences between groups in terms of the level of quantitative traits. The level of statistical significance was taken as p
<0.05. Statistical analysis was performed using the STATISTICA 6.0 software.
Results
Quality assessment of ChMMSC
The number of cells in the CHMMSC samples was 2 * x106 / ml, which confirms the proper quality of the product obtained by us in the process described in the previous report.
Macroscopic examination
Examination of the lower extremities did not reveal signs of wound infection or synovitis of the knee joint in both the study and control groups. when examining the articular surfaces of the thigh in the study group 1 and 2, complete filling of the defect area with tissue visually similar to cartilage was visualized. good integration of the regenerate with the surrounding healthy articular surface was determined (Fig. 2 A, B,c).
in control group 1, filling of the defect area with fibrous tissue was observed. the surface of the regenerate was uneven; an impression was determined in its center (Fig. 2D). in one case, clear signs of the development of gonarthrosis were noted - deformity of the joint, damage to the surrounding cartilage defect (Fig. 2 E).
In control group 2, the area of the defect was filled with tissue similar to cartilaginous, but the surface of the regenerate remained uneven, bumpy, its edges were clearly distinguishable (Fig. 2F).
Microscopic examination
indicators of category values and final values on the O'Driscoll scale in the studied and control groups of animals are presented in the table.
Discussion
In our study, the number of cells in the CHMMSC samples was 2 * 106 / ml (0.754 * 106 / pl; 2.21 * 106 / ml), which confirms the proper quality of the product obtained by us in the process of MMSC differentiation in the chondral direction. The use of a one-step differentiation protocol to obtain CHMMSC allowed us to avoid significant material costs associated with the use of specialized equipment and consumables.
The study did not reveal a single case of purulent inflammation of the wound in the postoperative period or any other complications, which makes the intra-ar-ticular administration of CHMMSC a safe technique.
11 weeks after the formation of the defect, the study group 1 (full-thickness osteochondral defect) was characterized by significantly higher indicators of the categories "Tissue morphology", "staining of the intercellular matrix", "Structural integrity", "thickness of the formed cartilage", "Cluster of chondrocytes", "severity of inflammation "(P <0.05) and the final value of the O'Driscoll scale (p <0.01) compared with the control. the area of the defect in animals of this group was filled mainly with hyaline cartilage and fibrocartilaginous tissue. Chondrocytes formed isogenic groups, and the staining of the extracellular matrix was close to normal. The regenerate is well integrated with the surrounding healthy cartilage. inflammation was moderately expressed (Fig. 3 A, B).
In control group 1, the regenerate was mainly represented by fibrous tissue with fibrocartilage inclusions. defined clear boundaries between the newly formed tissue and the surrounding articular surface. the surface of the regenerate itself was uneven. in this group, signs of pronounced inflammation were observed (Fig. 3 D, E).
The study and control group 2 were characterized by good filling of the defect area mainly with hyaline cartilage tissue (Fig. 3 c, F). The regenerates were firmly integrated with the surrounding articular surfaces. in control group 2, more pronounced processes of inflammation and hypocellularity were observed, which was expressed in significantly lower indicators of the corresponding categories of the scale (p <0.05). also the study group 2 was characterized by higher final values on the O'Driscoll scale (p <0.05) compared to the control.
Table 1.
The study showed that intra-articular injection of CHMMSC can significantly improve both the quantitative (degree of filling the defect) and qualitative (the integrity of the structure of the regenerate, the nature of its surface, etc.) characteristics of the newly formed cartilaginous tissue in the defect zone, which was especially evident in the group animals that developed an osteochondral full-thickness defect of the articular surface. regeneration in this area occurred with the participation of bone marrow mesenchymal stem cells. our data are consistent with the results of studies by A. Mishra et al. [14], who showed that CHMMSC accelerates the proliferation of mesenchymal stem cells and
categories Values of indicators of the O'Driscoll scale me (25th percentile, 75th percentile)
full thickness defect partial defect
study group 1 (intra-articular introduction of CHMMSC) control group 1 (no treatment) p study group 2 (intra-articular introduction of CHMMSC) control group 2 (no treatment) p
morphology of tissue 3 (2; 3) 2 (1; 2) 0,032 2 (2; 3) 2 (2; 2) 0,095
staining intercellular matrix staining 2 (2; 2) 1 (1; 2) 0,049 2 (2; 2) 2 (1; 2) 0,134
surface character 2 (2; 2) 2 (2; 2) 0,317 2 (2; 2) 2 (2; 2) 0,317
Integrity of structures 2 (2; 2) 1 (1; 1) 0,014 2 (2; 2) 1 (1; 2) 0,049
further promotes their chondrogenic differentiation. combined use of CHMMSC and these surgical inter-
Since osteoperforative techniques are based on the ventions can significantly improve the results of treat-
stimulation of regeneration in the area of cartilage de- ment of patients with articular cartilage defects. feet due to activation of mesenchymal stem cells, the
FIG. 3. Area offull-thickness (A, E, B, E) and non-full-thickness (r, ff) defect in the cartilage of the knee joint of a rabbit (2 weeks after the formation of the defect). A, E, H - study group; r, ff, E - control group. H - area of intact hyaline cartilage. P - regenerate. Staining with hematoxylin and eosin. Uv. X100, x200
A decrease in the severity of both vascular and cellular elements of inflammation in tissues with intra-ar-ticular administration of CHMMSC suggests its antiinflammatory effect, which also correlates with the results of studies by other authors [15].
Conclusions
1. Intra-articular administration of CHMMSC has a stimulating effect on the regeneration of hyaline cartilage in both full-thickness and partial-thickness defects.
2. The use of CHMMSC makes it possible to improve the quantitative and qualitative characteristics of the cartilaginous tissue newly formed in the process of regeneration in the zone of a full-thickness osteochondral defect of the articular surface.
3. CHMMSC, when administered intra-articular, has an anti-inflammatory effect. This method of CHMMSC administration is a safe method for treating traumatic defects of articular cartilage.
References
1. Widuchowski W. Articular cartilage defects: study of 25,124 knee arthroscopies / W. Widuchowski,
J. Widuchowski, T. Trzaska // Knee. — 2007 Jun. -Vol. 14, N 3. - P. 177-82.
2. Newman A. P. Articular cartilage repair / A. P. Newman // Am J Sport Med. - 1998 Mar-Apr. - Vol. 26, N 2. - P. 309-24.
3. Focal cartilage defects in the knee impair quality of life as much as severe osteoarthritis: a comparison of knee injury and osteoarthritis outcome score in 4 patient categories scheduled for knee surgery / S. Heir [et al.] // Am J Sports Med. - 2010. - Vol. 38, N 2. - P. 231-37.
4. Management of Focal Chondral Lesion in the Knee Joint / S. S. Seo [et al.] // Knee Surg Relat Res. -2011 Dec. - Vol. 23, N 4. - P. 185-96.
5. Adult equine bone marrow stromal cells produce a cartilage-like ECM mechanically superior to animal- matched adult chondrocytes / P. W. Kopesky [et al.] // Matrix Biol. - 2010 Jun. - Vol. 29, N 5. - P. 42738.
6. Microfracture for treatment of knee cartilage defects in children and adolescents / G. M. Salzmann [et al.] // Pediatr Rep. - 2012 Apr 2. - Vol. 4, N 2. - P. e21.
